ICAM-1 antisense for the treatment of inflammation and pain in a rectal stump

ABSTRACT

This invention relates to a composition comprising an antisense oligonucleotide that down-regulates intracellular adhesion molecule-1 (ICAM-1) for use in treating inflammation, pain and/or discharge in a rectal stump.

This invention relates to a composition comprising an antisenseoligonucleotide that down-regulates intracellular adhesion molecule-1(ICAM-1) for use in treating inflammation, pain and/or discharge in arectal stump.

Patients suffering from disorders of the intestine may have to undergosurgery to remove a section of the intestine or the entire intestine.For example, this can be necessary in patients with colon cancer,diverticulitis and inflammatory bowel diseases such as ulcerativecolitis or Crohn's disease.

In general, a surgeon will try and minimise the extent of the surgeryand may leave the rectal stump intact. However, the rectal stump canbecome aggravated and inflamed, be very painful and/or discharge mucusand/or blood. It is believed a trigger is the absence of stools and aresulting change in the local environment and an absence of faecalbacterial flora.

In some cases, steroids, 5-aminosalicyclic acid (5-ASA) orimmunomodulators are used to treat the aggravated rectal stump but anumber of patients are resistant to these drugs and therefore analternative approach for alleviating an aggravated rectal stump isrequired.

An ICAM-1 antisense oligonucleotide comprising the nucleic acid sequenceof SEQ ID NO: 1 has previously been used to treat ulcerative colitis(UC) and pouchitis, both of which are conditions of a continuousintestine (not a blind ended intestine).

ICAM-1, a member of the immunoglobulin (Ig) superfamily, is an inducibletransmembrane glycoprotein constitutively expressed at low levels onvascular endothelial cells and on a subset of leucocytes (Dustin et al.,J. Immunol, 137:245-54, 1986; Rothlein et al, J. Immunol., 137:1270-4,1986; Simmons et al, Nature, 331:624-7, 1988). SEQ ID NO:1 is a 20-basephosphorothioate oligodeoxynucleotide designed to specifically hybridizeto a sequence in the 3′-untranslated region of the human ICAM-1 mRNA.Studies strongly suggest that the ICAM-1 antisense oligonucleotidefunctions by specifically binding to the ICAM-1 mRNA resulting incleavage of the mRNA by the enzyme RNaseH1 (Crooke, Biochim. Biophys.Acta., 1489:31-44, 1999), one of an ubiquitous family of RNaseHnucleases.

It has been surprisingly found, by chance, that a composition comprisingan oligonucleotide having a sequence comprising SEQ ID NO: 1 isextremely effective for reducing inflammation, pain and/or discharge inthe rectal stump.

According to a first aspect of the invention, there is provided acomposition comprising an oligonucleotide comprising the nucleic acidsequence of SEQ ID NO:1 and hydroxypropyl methylcellulose for use intreating inflammation, pain and/or discharge in a rectal stump.

SEQ ID NO:1 is as follows: 5′-gcccaagctg gcatccgtca-3′

In one embodiment, the composition comprises an oligonucleotideconsisting of the nucleic acid sequence of SEQ ID NO:1.

The oligonucleotides in accordance with this invention preferablycomprise from about 20 to about 80 nucleic acid base units. It is morepreferred that such oligonucleotides comprise from about 20 to 50nucleic acid base units, still more preferred to have from about 20 to30 nucleic acid base units, and most preferred to have from about 20 to22 nucleic acid base units. As will be appreciated, a nucleic acid baseunit is a base-sugar combination suitably bound to an adjacent nucleicacid base unit through phosphodiester or other bonds. One skilled in theart will understand that about 20 to about 80 nucleic acid base unitsincludes 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79 or 80 nucleobase units.

In a further embodiment, the composition comprises a fragment of SEQ IDNO:1, wherein the fragment is at least 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18 or 19 nucleotides in length. The fragment can hybridise to asequence in the 3′-untranslated region of the human ICAM-1 mRNA. Thefragment can hybridise under moderate or stringent conditions withnucleotides ‘cctgacg gatgccagct tgg’ (SEQ ID NO:2). Fragments include‘cccaagctg gcatccgtca’ (SEQ ID NO:3), ‘gcccaagctg gcatccgtc’ (SEQ IDNO:4) and ‘gcccaagctg gca’ (SEQ ID NO:5). “Stringency” of hybridizationreactions is readily determinable by one of ordinary skill in the art,and generally is an empirical calculation dependent upon probe length,washing temperature, and salt concentration. In general, longer probesrequire higher temperatures for proper annealing, while shorter probesneed lower temperatures. Hybridization generally depends on the abilityof denatured DNA to reanneal when complementary strands are present inan environment below their melting temperature. The higher the degree ofdesired homology between the probe and hybridisable sequence, the higherthe relative temperature which can be used. As a result, it follows thathigher relative temperatures would tend to make the reaction conditionsmore stringent, while lower temperatures less so. For additional detailsand explanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

As herein defined, “Stringent conditions” or “highly stringencyconditions”, may be identified by those that: (1) employ low ionicstrength and high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulphate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3)employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mMsodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt'ssolution, sonicated salmon sperm DNA (50 [mu]g/ml), 0.1% SDS, and 10%dextran sulphate at 42° C., with washes at 42° C. in 0.2×SSC (sodiumchloride/sodium citrate) and 50% formamide at 55° C., followed by ahigh-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.

“Moderately stringent conditions” may be identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual, New York: ColdSpring Harbor Press, 1989, and include the use of washing solution andhybridization conditions (e.g., temperature, ionic strength and % SDS)less stringent that those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulphate, and 20 mg/ml denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37-50° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

As used herein, conditions of moderate or high stringency can be readilydetermined by those having ordinary skill in the art based on, forexample, the length of the DNA. The basic conditions are set forth bySambrook et al. Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1,pp. 1.101-104, Cold Spring Harbor Laboratory Press, (1989).

The oligonucleotide can be modified to comprise at least onephosphorothioate linkage. Phosphorothioate modification of theoligonucleotide, by substituting a sulfur molecule for a non-bridgingoxygen molecule in each phosphodiester linkage, significantly increasesexonuclease resistance relative to unmodified DNA and prolongs the drughalf life (Geary et al., Anti-Cancer Drug Design, 12:383-94, 1997).Phosphorothioate oligonucleotides are only minimally antigenic,non-cytotoxic and well tolerated, and their pharmacokinetic andpharmacodynamic properties are well characterized (see e.g., Butler etal., Lab. Invest, 77:379-88, 1997; Mirabelli et al., Anti-Cancer DrugDes., 6:647-61, 1991).

In addition to phosphorothioate backbone modifications, a number ofother possible backbone, sugar and other modifications are well known tothose skilled in the art.

The rectal stump that benefits from being treated is an aggravatedrectal stump. References to “aggravated rectal stump” herein mean arectal stump that is inflamed, painful and/or discharges blood and/ormucous. Aggravation of the rectal stump is believed to be caused by anabsence of stools, which results in an absence of faecal flora. Theinflammation, pain and/or discharge can be symptoms of diversion colitis(also known as dysfunctional colitis). Accordingly, the composition canbe for use in treating a subject with diversion colitis.

The composition for the use of the invention is particularly useful asthe alternative is further surgery to remove the rectal stump, which isclearly very invasive for the patient and therefore traumatic,particularly as the patient will already have undergone significantsurgery. Furthermore, there is a risk of death associated with thefurther surgery.

A rectal stump is formed when the rectum is closed and excluded fromfaecal transit after removal of the colon. The rectal stump can be aclosed ended rectal stump or the upper end can exit to the lower part ofan abdominal incision or as a mucous fistula in the left iliac fossa. Inall cases, the rectum has been surgically severed from thegastro-intestinal tract.

In one embodiment, the composition is for use in treating a subject thatdoes not suffer from colitis, particularly UC, and/or pouchitis. In afurther embodiment, the subject has never suffered from colitis,particularly UC, and/or pouchitis.

The composition can be formulated for rectal administration.Compositions for rectal administration include solutions, such as enemasand suppositories, and emulsions or foams. Absorption promotingadjuvants can be included with the composition.

Formulations for the rectal delivery of pharmaceutical compositions arewell known to those skilled in the art. The selection of a specificformulation is based on considerations well known to those skilled inthe art. Detailed formulations are presented in U.S. Pat. Nos. 6,096,722and 6,747,014 both incorporated herein by reference.

The composition may be for use in a patient that has a rectal stump ofless than 25 cm, 20 cm, 15 cm, 10 cm, 9 cm, 8 cm, 7 cm, 6 cm or lessthan 5 cm.

The composition comprising an oligonucleotide comprising the nucleicacid sequence of SEQ ID NO:1 and hydroxypropyl methylcellulose may beadministered alone but can also be combined with another pharmaceuticalagent.

In a preferred embodiment, the subject is human.

The precise dose of the oligonucleotide will depend upon a number offactors, including the severity of the inflammation, pain and/ordischarge. The composition is preferably administered to an individualin a “therapeutically effective amount”, this being sufficient to showbenefit to the individual.

The actual amount administered, and rate and time-course ofadministration, will depend on the nature and severity of what is beingtreated. Prescription of treatment, e.g. decisions on dosage etc, isultimately within the responsibility and at the discretion of generalpractitioners and other medical doctors, and typically takes account ofthe disorder to be treated, the condition of the individual patient, thesite of delivery, the method of administration and other factors knownto practitioners.

For example, in one embodiment, a suitable dose may be 60 ml/240 mg, 70ml/240 mg, 80 ml/240 mg, 90 ml/240 mg, 100 ml/240 mg, 30 ml/120 mg, 60ml/120 mg, 70 ml/120 mg, 80 ml/120 mg, 90 ml/120 mg, 100 ml/120 mg perdose, for example, per enema.

The composition may be administered once, twice, three or four times aday or periodically.

The composition can be administered for 3, 4, 5, 6, 7, 8 or more weeks.A peak response is achieved 8, 9, 10, 11, 12 weeks or more after thetreatment commences. The patient can be in remission for 4-12, 6-12,6-18 or more including 16-24 months after treatment.

The composition may be in respect of existing aggravation, inflammation,pain and/or discharge of the rectal stump or may be prophylactic(preventative treatment). Treatment may include curative, alleviation orprophylactic effects.

More specifically, treatment includes “therapeutic” and “prophylactic”and these types of treatment are to be considered in their broadestcontext. The term “therapeutic” does not necessarily imply that asubject is treated until total recovery. Similarly, “prophylactic” doesnot necessarily mean that the subject will not eventually contract adisease condition.

Accordingly, therapeutic and prophylactic treatment includesamelioration of the symptoms of a particular condition or preventing orotherwise reducing the risk of developing a particular condition. Theterm “prophylactic” may be considered as reducing the severity or theonset of a particular condition. “Prophylactic” also includes preventingreoccurrence of a particular condition in a patient previously diagnosedwith the condition. “Therapeutic” may also reduce the severity of anexisting condition.

According to a second aspect of the invention, there is provided amethod of treating inflammation, pain and/or discharge in the rectalstump in a subject, comprising administering a composition comprising anoligonucleotide comprising the nucleic acid sequence of SEQ ID NO:1 andhydroxypropyl methylcellulose to the subject.

In one embodiment, the subject is in need of such treatment or canbenefit from such treatment.

A therapeutically effective amount of the oligonucleotide isadministered to the subject.

The term “therapeutically effective amount” as used herein in thecontext of treating aggravated rectal stump means an amount capable ofreducing inflammation, pain and/or discharge relative to theinflammation, pain and/or discharge experienced by the subject beforethe composition of the invention is administered.

The term ‘treatment’ is used herein to refer to any regimen that canbenefit a human

Preferred features for the second and subsequent aspects of theinvention are as for the first aspect mutatis mutandis.

The invention will now be further described by way of reference to thefollowing Example, which is provided for the purposes of illustrationonly and are not to be construed as being limiting on the invention.

EXAMPLE 1

This patient had a sub-total colectomy and ileostomy for activeulcerated colitis and underwent emergency surgery.

Surgery was performed as the patient was not responding to normalmedical interventions for this condition.

Post Operative—patient presented with constant pain and a post rectaldischarge of bleeding and mucus anything between 17-19 times over aperiod of 24 hours. He had rectal 5ASA to try and control the discharge.In July 2011 symptoms were worsening. The gastroenterologist wasreluctant to use Aziathioprine as it had previously not been effective.The other option would have been use of Biologicals but theGastroenterologist was reluctant to prescribe due to systemic effects.

He underwent proctoscopy on 19^(th) August and commenced a compositioncomprising SEQ ID NO:1 on 22^(nd) August.

He came back for review on the 4^(th) October. At that review he hadreduced his post rectal discharge to blood only and to about 8-9 times aday. There was no pain and no mucus discharge.

The Specialist Nurse spoke to him on the 19 Jan. 2012 and he dischargehad reduced to 3-6 times over a 24 hour period.

The patient feels that his quality of life has been significantlyimproved and that he has benefited greatly from the course of acomposition comprising SEQ ID NO:1.

The invention claimed is:
 1. A method of treating inflammation, painand/or discharge in a rectal stump in a subject, comprisingadministering a composition comprising an oligonucleotide comprising thenucleic acid sequence of SEQ ID NO: 1 and hydroxypropyl methylcelluloseto a subject having a rectal stump.
 2. The method of claim 1, whereinthe composition is formulated as an enema, suppository, emulsion or foamand the administering is for rectal administration.
 3. The method ofclaim 1, wherein the rectal stump exhibits mucosal and/or blooddischarge.
 4. The method of claim 1, wherein the oligonucleotidecomprises at least one phosphorothioate linkage.
 5. The method of claim1, wherein the rectal stump is 25 cm or less length.
 6. The method ofclaim 1, wherein the inflammation, pain and/or discharge are symptoms ofdiversion colitis.
 7. The method of claim 1, wherein the composition isadministered in an amount effective to reduce inflammation, pain and/ordischarge in a rectal stump.
 8. The method of claim 7, wherein theinflammation, pain and/or discharge are symptoms of diversion colitis.